Pig Heart Lipoamide Dehydrogenase Research Articles

Ion pair formation in pig heart lipoamide dehydrogenase: rationalization of pH profiles for reactivity of oxidized enzyme with dihydrolipoamide and 2-electron-reduced enzyme with lipoamide and iodoacetamide.

Ion pair formation in pig heart lipoamide dehydrogenase: rationalization of pH profiles for reactivity of oxidized enzyme with dihydrolipoamide and 2-electron-reduced enzyme with lipoamide and iodoacetamide.

Spectral evidence for a flavin adduct in a monoalkylated derivative of pig heart lipoamide dehydrogenase.

A derivative of the flavoprotein pig heart lipoamide dehydrogenase has been described recently (Thorpe, C., and Williams, C.H. (1976) J. Biol. Chem. 251, 3553-3557), in which 1 of the 2 cysteine residues generated on reduction of the intrachain active center disulfide bridge is selectively alkylated with iodoacetamide. This monolabeled enzyme exhibits a spectrum of oxidized bound flavin. The addition of 1 mM NAD+ to this derivative at pH 8.3 causes a decrease in absorbance of approximately 50% at 448 nm, with a concomitant increase at 380 nm. These spectral changes are complete within 3 ms and are reversible. NAD+ titrations generate isosbestic points at 408, 374, and 327 nm; allowing values for the apparent dissociation constant for NAD+ and the extent of bleaching at infinite ligand to be obtained from double reciprocal plots. Between pH 6.1 and 8.8, the apparent KD decreases from 320 to 35 muM, whereas the extrapolated delta epsilon 448 values remain approximately constant at 1/2 epsilon 448. Direct measurement of NAD+ binding by gel filtration at pH 8.8 indicates that the spectral changes are associated with a stoichiometry of 1.2 mol of NAD+ bound/2 mol of FAD. The modified protein is a dimer containing 1 FAD and 1 alkylated cysteine residue/subunit; the native enzyme is also dimeric. The visible spectrum of the species absorbing at 380 nm, approximated by correction for the residual oxidized FAD, shows a single maximum at 384 nm, epsilon 384 = 8.7 mM-1cm-1. Comparison of this spectrum with that of model compounds of known structure suggests that it may represent a reversible covalent flavin adduct induced on binding NAD+.

Differential reactivity of the two active site cysteine residues generated on reduction of pig heart lipoamide dehydrogenase.

Reduction of the active center disulfide bond in the flavoprotein pig heart lipoamide dehydrogenase generates two sulfur moieties which are chemically inequivalent in the 2-electron reduced form of the enzyme. Thus 1 cysteine residue is at least 13-fold more reactive than its partner toward iodoacetamide at pH 7.6. This selectivity was demonstrated by reaction of the 2-electron reduced enzyme with a low concentration of iodo[1-14C]acetamide under anaerobic conditions. The formation of a monolabeled derivative is accompanied by the reappearance of a spectrum of oxidized bound flavin, clearly different from that of the native enzyme. Alkylation of the remaining cysteine residues with iodo[12C]acetamide enabled the isolation of a tryptic version of the active center disulfide peptide. A single chymotryptic cleavage between the 2 alkylated cysteine residues generated a cationic and an anionic fragment containing 7% and 93% of the radioactivity of the purified tryptic peptide, respectively. The monolabeled derivative is catalytically inactive toward reduced or oxidized lipoamide, but is approximately 2-fold better as a transhydrogenase than the native protein using NADH and acetylpyridine adenine dinucleotide as substrates. Anaerobic titration with NADH leads to reduction of the flavin with concomitant formation of long wavelength absorption of low intensity. No intermediate reduced states were detected in this titration analogous to the red 2-electron form observed with the native enzyme. Similarly, intermediates during reduction of the enzyme by 1 eq of dithionite have not been detected.

Interaction of hydrophobic probes with the apoenzyme of pig heart lipoamide dehydrogenase.

The interaction of hydrophobic probes, 8-anilinonaphthalene-1-sulfonate (ANS) and 4-benzoylamido-4'-aminostilbene-2, 2'-disulfonate (MBAS), with pig heart lipoamide dehydrogenase [NADH: lipoamide oxidoreductase, EC 1.6.4.3] was investigated. When ANS or MBAS was mixed with the apoenzyme of lipoamide dehydrogenase, the fluorescence quantum yield, of each dye was enhancedd markedly and the emission maxima concurrently shifted to the blue. The quantum yield, 0.038, of ANS bound to the apoenzyme, calculated from the corrected emission spectrum, was eight times higher than that in buffer solution, and the value, 0.0090, for bound MBAS was eighteen times higher than that in buffer solution. Moreover, the absortion bands of both ANS and MBAS shifted to the red upon binding with the apoenzyme. A general feature of the absorption spectra of these dyes observed on changing the solvent from polar to apolar was a red shift of the absorption bands. These results indicate that ANS or MBAS bound to the apoenzyme of lipoamide dehydrogenase is situated in a hydrophobic region of the apoenzyme molecule. It was found that 2 moles of each dye was bound per mole of the apoenzyme, which contains two polypeptide chains. The dissociation constants for the ANS- and MBAS-apoenzyme complexes were estimated to be 1.03X10(-5) and 1.54X10(-5) M, respectively. The enhanced fluorescence of both dyes bound to the apoenzyme decreased linearly upon adding FAD and disappeared at about 2 moles of FAD per mole of the apoenzyme. This suggests that both ANS and MBAS were displaced from their binding sites on the apoenzyme by FAD. The protein fluorescence spectrum of the apoenzyme had a maximum at 352 nm, which was blue-shifted by 6 nm from that of tryptophan in the buffer. Upon binding ANS or MBAS, the maximum of the protein fluorescence of the apoenzyme returned to 350 nm for the holoenzyme, and the fluorescence intensity decreased. Thus, the conformation around some tryptophan residues was affected by the binding of the dyes. When guanidine hydrochloride (GuHCl) was added to the ANS-apoenzyme complex solution, the enhanced fluorescence due to the bound ANS decreased and the emission maximum concurrently shifted to the red. Further, the maximum of the protein fluorescence of the apoenzyme shifted to the red, indicating the exposure of some tryptophan residues buried in an apolar region of the apoenzyme. Thus the binding of ANS to the apoenzyme was inhibited by protein denaturation due to GuHCL. In contrast, the holoenzyme of lipoamide dehydrogenase did not bind ANS or MBAS at all.

Modification of pig heart lipoamide dehydrogenase by cupric ions.

The insertion of a second disulfide bridge into native pig heart lipoamide dehydrogenase, requires two Cu-2+ ions for each catalytic center inactivated under anaerobic conditions. During inactivation, both metal atoms become reducible by their juxtaposition to the two participating cysteine residues and may be removed as the Cu+-chelates of neocuproine and bathocuproinesulfonate, leaving an additional disulfide bridge on the protein. Inactivation does not require the presence of oxygen, but when substoichiometric levels of copper are used under aerobic conditions the slow regeneration of Cu-2+ becomes rate-limiting. The course of aerobic inactivation is markedly biphasic at 0 degrees using 2 Cu-2+/FAD, with 30% of the total change completed rapidly, followed by a much slower phase. Both the extent of the fast phase and the rate of the second phase are enhanced by increasing levels of Cu-2+, but are relatively unaffected when the Cu-2+/FAD ratio is maintained at 2 and the protein concentration is varied. The enzyme affords several binding sites for Cu-2+ at pH 7.8, and it is suggested that competition between these sites during the initial statistical distribution of metal ions may explain this biphasic behavior.

The sequence of amino acid residues around the oxidation-reduction active disulfide in yeast glutathione reductase.

A 14-residue peptide containing the oxidation-reduction active cystine residue from yeast glutathione reductase has been isolated from proteolytic digests of the enzyme in which the free sulfhydryl groups had been reacted with N-ethylmaleimide. The sequence of this disulfide-containing peptide was found to be:(see article). The sequence was highly homologous with the active cystine regions in Escherichia coli and pig heart lipoamide dehydrogenase. The sequences of three of the postulated four thiol-containing regions of the enzyme are also presented, as well as evidence supporting the view that the enzyme is composed of two identical subunits.

Isolation, characterization and partial sequencing of cystine and thiol peptides of pig heart lipoamide dehydrogenase

Pig heart lipoamide dehydrogenase (EC 1.6.4.3) contains ten half-cystines (as cysteic acid) per mole of enzyme bound FAD. Two of these are linked in an intrachain cystine which acts in concert with the flavin during catalysis. A peptic peptide containing this active center disulfide has been isolated and shown to have the sequence: Glu-Thr-Leu-Gly-Gly-Thr-Cys-Leu-Asn-Val-Gly-Cys-Ile-Pro-Ser (Lys, Ala, Leu). The enzyme also contains seven titratable thiols when either 5,5′-dithiobis (2-nitrobenzoic acid) or iodoacetate is used as titrant. Tryptic peptides containing alkylated thiols have been isolated and characterized by amino acid composition and by their positions in two-dimensional chromatography-electrophoresis. On the basis of map position and composition, the peptides containing thiols can be distinguished from one another. The results are compared with recent data of Brown and Perham (Brown, J. P. and Perham, R. N. (1974) Biochem. J. 138, 505–512) on the compositions and partial sequences of tryptic chymotryptic peptides containing half-cystines. The combined data associate nine of the ten half-cystines with unique compositions.

Identification of the thiol residues involved in modifications of pig heart lipoamide dehydrogenase by cupric ion and by iodoacetamide

The thiol residues involved in two previously described modifications of heart lipoamide dehydrogenase (NADH:lipoamide oxidoreductase, EC 1.6.4.3) have been identified by comparison of peptide maps of unmodified and modified enzymes. Two thiols and one methionine react when native enzyme is alkylated in concentrated iodoacetamide, with the accompanying loss of enzymatic activity and an NAD-binding site. NAD protects the more slowly reacting thiol from akylation, and the NAD-protected enzyme is active and retains its NAD-binding site. Loss of the binding site, and loss of activity are associated with the alkylation of two neutral thiol peptides which may represent alternative versions of a single thiol region in the enzyme. Treatment of native enzyme with cupric ion results in the rapid oxidation of two thiols to a disulfide bond and loss of NADH-lipoamide reductase activity. We have determined that thiol residues in the cationic peptide and one of the anionic peptides are involved in the disulfide bond formed by cupric ion. Since the cationic peptide contains two histidyl residues, it is proposed that it is the initial site of binding of cupric ion, prior to the oxidation of the thiol residues to a disulfide bond.

Effects of low concentrations of guanidine hydrochloride on pig heart lipoamide dehydrogenase.

Effects of low concentrations of guanidine hydrochloride on pig heart lipoamide dehydrogenase.

Escherichia coli B cobalamin methyltransferase: Ability of diaphorases and lipoamide dehydrogenases to function as reducing agents

An FAD containing diaphorase and lipoamide dehydrogenase were purified as by-products in the isolation of cobalamin methyltransferase from extracts of Escherichia coli B. In the presence of 1,4-dithiothreitol (DTT) and/or NADH these two flavoproteins, as well as pig heart lipoamide dehydrogenase, were shown to replace substantially the requirement for free FMNH 2 (or FADH 2) by the bacterial methyltrausferase. Assay systems containing each of these three flavoproteins, respectively, differed in their dependencies on DTT and NADH. The differences were attributed, in part, to the ability of E. coli B lipoamide dehydrogenase to use DTT as a substrate in comparison to the bacterial diaphorase and pig heart lipoamide dehydrogenase. In addition, a preliminary incubation of the cobalamin protein with DTT markedly increased its activity in the presence of two of the NADH-reduced flavoproteins. For each of the three flavoproteins 1 mμmole of added, bound FAD could be shown to support the synthesis of 10 mμmoles of 14CH 3−-methionine. With the use of NADH + iodoacetamide-treated pig heart lipoamide dehydrogenase, it was demonstrated that both the semiquinoid (FADH·) state and the fully reduced (FADH 2) form of a single flavoprotein can function as reductants for the cobalamin enzyme.

Optical Activity and Conformation Studies of Pig Heart Lipoamide Dehydrogenase

Abstract Flavin adenine dinucleotide has been dissociated from pig heart lipoamide dehydrogenase in the presence of 1.5 m guanidine hydrochloride. Measurements of the circular dichroism on the apoenzyme show evidence of a large change in conformation, based on the molar ellipticity in the region 240 to 200 mµ. When FAD is removed from the holoenzyme in guanidine, but in the presence of approximately equimolar FMN, no change appears in the circular dichroism of the apoenzyme in the 200 mµ region. The ultraviolet absorption of the apoprotein indicates that no FMN remains bound to the apoenzyme. Either FMN stabilizes the protein in the presence of guanidine or, after the loss of FAD, promotes the return of a conformationally different species to the native form of the enzyme. Lipoamide dehydrogenase activity is reconstituted in the presence of FAD and dithiothreitol.

Intramitochondrial distribution of multiple forms of pig heart lipoamide dehydrogenase

Intramitochondrial distribution of multiple forms of pig heart lipoamide dehydrogenase